Skip to main content

Isolation and full-length genome analysis of mosquito-borne Manzanilla virus from Yunnan Province, China



There have been four strains on Manzanilla virus (MANV) identified to date. Here, we identify a novel MANV strain (DHL10M107) isolated from Culex tritaeniorhynchus Giles mosquitoes from Ruili city, Dehong prefecture, Yunnan Province, in the People’s Republic of China.


The DHL10M107 L, M and S genes were sequenced at the nucleotide and deduced amino acid levels. The L, M and S gene sequences of DHL10M107 clustered with the MANV strains VN04-2108, TRVL3587, SA An 4165, and AV 782. DHL10M107 was most closely related to VN04-2108. Nucleotide homology ranged between 96 and 99% between DHL10M107 and VN04-2108. In terms of amino acid homology, all of the amino acid differences were in the L (96.3% homologous) and M (97.7% homologous) fragments.


DHL10M107 is likely a MANV isolated from mosquitos in the Yunnan Province. This is the first reported isolation of MANV in mainland China.


Manzanilla virus (MANV) belongs to the Simbu serogroup of the genus Orthobunyavirus of family Bunyaviridae [13]. MANV is a single-stranded negative sense RNA virus that contains three RNA fragments: large (L), medium (M) and small (S). Four strains of MNV have been isolated from to date. Anderson et al. were the first group to isolate MANV (TRVL3587) [1] from the blood of a Howler monkey (Alouatta seniculus insularis) in Trinidad in 1954. More recently, in 2004 Bryant et al. isolated a MANV strain identified as the Cat Que virus (VN04-2108) from mosquitoes (Culex sp.) in Vietnam [2]. In 2014, Ladner et al. [3] reclassified two viruses as MANV strains, the Ingwavuma virus (SA An 4165) isolated from a South African spectacled weaver (Hyphanturgus ocularis) in 1959 [4], and the Mermet virus (AV 782) isolated from a North American purple martin (Progne subis) in the United States in 1964 [5].

Yunnan Province is located in southwest China, adjacent to Myanmar, Laos and Vietnam. Ecologically, Yunnan Province supports the distribution of vector mosquitoes and the transmission of mosquito-borne viruses [6, 7]. The Japanese encephalitis virus (Genus Flavivirus, Family Flaviviridae), Dengue virus (Genus Flavivirus, Family Flaviviridae), Chikungunya virus (Genus Alphavirus, Family Togaviridae), Sindbis virus (Genus Alphavirus, Family Togaviridae), Getah virus (Genus Alphavirus, Family Togaviridae), Batai virus (BATV) (Genus Orthobunyavirus, Family Bunyaviridae), Banna virus (Genus Seadornavirus, Family Reoviridae), Yunnan orbivirus (Genus Obivirus, Family Reoviridae), and several other viruses have been isolated from human patients and from mosquitoes collected in Yunnan Province [816]. This study aimed to further investigate the distribution of mosquito-borne viruses in Yunnan Province.

In August 2010, mosquitoes were collected using the Kongfu Xiaoshuai trap (Wuhan Jixing Environmental Protection, Scientific, and Technological LLC) from cattle barns in the suburb of Ruili city, Dehong prefecture, Yunnan Province, People’s Republic of China. A total of 425 mosquitoes representing 10 species in four genera were collected. Of these, 318 (74.82%) were Culex tritaeniorhynchus Giles and the remaining 107 were from the other nine species (25.18%). The mosquito samples were sorted by species into 15 pools and ground together. The supernatant was used to inoculate Baby hamster kidney (BHK-21) cells to isolate any viruses present as previously described [15]. One of the pools containing C. tritaeniorhynchus (DHL10M107) caused significant cytopathic effects (CPE) in BHK-21 cells, characterized by the cells shrinking, rounding, and eventually floating.

The viral RNA was extracted from the culture supernatant of the DHL10M107 isolate, using the QIAamp viral RNA mini kit (Qiagen, Valencia, CA, USA) per the manufacturer’s instructions. cDNA was prepared with Ready-To-Go You-Prime First-Strand Beads™ (American Amersham Pharmacia Biotech, Piscataway, NJ, USA), and amplified with MANV specific primers (see Additional file 1: Table s1). The reactions contained 1 µL cDNA template, 10 µL 2 × GCI Buffer, 0.5 µL 2.5 mM dNTP, 0.4 µL each of the upstream and downstream primers (10 mM), 0.4 µL ExTaq enzyme (5 U/µL), and 7.3 µL deionized water. The reaction conditions were: pre-denaturing at 95°C for 3 min, followed by 32 cycles of 95°C for 30 s, 58°C for 40 s, and 72°C for 1–2 min, and extension at 72°C for 10 min. The amplification was assessed using 2 µL of the product for 1% agarose gel electrophoresis. The whole genome sequence of the DHL10M107 strain was obtained (Beijing Liuhe Genomics Technology Co., Ltd. Shenzhen Branch). The L, M and S sequences of DHL10M107 strain were submitted to GenBank under accession nos. KP016012-KP016014. The sequence fragments were spliced, edited and corrected using SeqMan (DNAstar software package) and the phylogenetic analysis and nucleotide sequence analysis were performed using ClustalX (version 1.8), MEGA5.0, DNAStar alignment package, and MegAlign software.

Phylogenetic analysis of the viral L and M gene sequences indicated that DHL10M107 clustered with MANV (VN04-2108) and an Oya virus (OYV) strain (SC0806) isolated from mosquitos in Sichuan province, China [17], although it was more closely related to VN04-2108 (Figure 1a, b). However, in terms of the S fragments, while DHL10M107 was in the same clade as VN04-2108 and SC0806, it formed a secondary clade, and the S fragments of VN04-2108 and SC0806 were more closely related (Figure 1c). Regardless of the genome fragment, DHL10M107 was closely related to three MANV strains (TRVL3587, SA An 4165, and AV 782; Figure 1). The OYV reported by Kono et al. [18] had a partial sequence for the S fragment in GenBank (accession no. AB075611). Similar to Figure 1c, the phylogenetic tree constructed using this sequence indicated AB075611, DHL10M107, VN04-2108, and SC0806 were in the same clade and had similar evolutionary relationships (Figure 1d).

Figure 1
figure 1

Phylogenetic trees based on the nucleotide sequences of DHL10M107 strains isolated from Yunnan Province, China. a L sequence, b M sequence, c S sequence and d partial Oya virus S sequence (365 bp). The black triangle indicates DHL10M107.

We then compared the homology of the open reading frame (ORF) and amino acid sequences in DHL10M107 to five other Orthobunyavirus strains (Table 1). There was minimal nucleotide and amino acid homology between DHL10M107 and Tahyna virus, BATV, and Oropouche virus (Table 1). In terms of nucleotide homology, the similarity of the DHL10M107 L fragment to VN04-2108 and SC0806 was 96.3 and 92.6%, respectively. The M fragment (VN04-2108: 97.7% and SC0806: 97.2%) and S fragment (VN04-2108: 98.7% and SC0806: 98.9%) had similarly high levels of homology. Amino acid homology was between 99 and 100% for DHL10M107, VN04-2108, and SC0806 (Table 1). All of the amino acid differences were in the L and M fragments. In the L fragment, DHL10M107 differed from VN04-2108 by six amino acids and SC0806 by 11 amino acids. In the M fragment the difference was four (VN04-2108) and five amino acids (SC0806; Table 2).

Table 1 Nucleotide and amino acid homology in the sequences from DHL10M107 and other Orthobunya viruses
Table 2 Sites of amino acid differences in L and M sequences of DHL10M107, VN04-2108 and SC0806

In conclusion, the high degree of similarity in the whole genome sequence between DHL10M107 and the four MANV strains in GenBank, but not other viruses in the Simbu serogroup, confirmed it was a MANV strain. This is the first report of MANV in mainland China. Currently, the records pertaining to MANV in the International Committee on Taxonomy of Virus (ICTV) [19] excludes Oya virus. Here, we identified two virus strains named OYV in GenBank. One was a partial OYV S fragment sequence (AB075611) isolated from the lungs of pigs thought to have Nipah virus infection in Malaysia [8]. The second strain was the entire sequence of OYV SC0806 (JX983192, JX983193 and JX983194) isolated from mosquitos in Sichuan province, China [7]. Phylogenetic analysis indicated that both OYV sequences clustered with the MANV strains. They were most closely related to VN04-2108 and DHL10M107, suggesting that OYV (SC0806 and AB075611) was also a member of the MANV complex.



large RNA genome fragment


medium RNA genome fragment


small RNA genome fragment


Manzanilla virus


Batai virus


baby hamster kidney cells-21


cytopathic effects


Oya virus


open reading frame


International Committee on Taxonomy of Virus


  1. Anderson CR, Spence LP, Downs WG, Aitken TH (1960) Manzanilla virus: a new virus isolated from the blood of a howler monkey in Trinidad, W I. Am J Trop Med Hyg 9:78–80

    CAS  PubMed  Google Scholar 

  2. Bryant JE, Crabtree MB, Nam VS, Yen NT, Duc HM, Miller BR (2005) Isolation of arboviruses from mosquitoes collected in northern Vietnam. Am J Trop Med Hyg 73(2):470–473

    PubMed  Google Scholar 

  3. Ladner JT, Savji N, Lofts L, da Rosa AT, Wiley MR, Gestole MC et al (2014) Genomic and phylogenetic characterization of viruses included in the Manzanilla and Oropouche species complexes of the genus Orthobunyavirus, family Bunyaviridae. J Gen Virol 95(Pt5):1055–1066

    Article  CAS  PubMed  Google Scholar 

  4. McIntosh BM, McGillivray GM, Dickinson DB (1965) Ingwavuma virus: an arbovirus isolated in South Africa. S Afr J Med Sci 30(3):67–70

    CAS  PubMed  Google Scholar 

  5. Calisher CH, Kokernot RH, De Moore JF, Boyd KR, Hayes J, Chappell WA (1969) Arbovirus studies in the Ohio-Mississippi Basin, 1964–1967. VI. Mermet: a Simbu-group arbovirus. Am J Trop Med Hyg 18(5):779–788

    CAS  PubMed  Google Scholar 

  6. Zhang YZ, Zhang HL, Gong ZD, Yang WH, Zhang YZ (2001) Studies on the mosquito communities and the geographical niche of border area in Yunnan Province. Chin J Vector Biol Control 12(1):28–32

    Google Scholar 

  7. Feng Y, Fu SH, Zhang HL, Petersen LR, Zhang BS, Gao XY et al (2013) High incidence of Japanese encephalitis, Southern China. Emerg Infect Dis 19(4):672–673

    Article  PubMed  Google Scholar 

  8. Zhang HL, Zi DY, Shi HF, Mi ZQ, Gong ZD, Zhang YZ et al (1999) Characterized distribution of Culex tritaeniorhynchus and their natural infection with Japanese encephalitis virus in Yunnan Province. Chin J Vector Biol Control 10(3):192–194

    Google Scholar 

  9. Deng SZ, Zhang HL, Li JM (2009) Distribution characteristics of mosquito and their natural infection with Japanese encephalitis virus in Yunnan Province. Chin J Vector Biol Control 20(4):344–348

    Google Scholar 

  10. Zhang HL, Zi DY, Gong ZD (1999) Epidemiological survey of dengue fever in Yunnan Province, China. Difangbing Tongbao 14(3):50–54

    CAS  Google Scholar 

  11. Zhang HL, Shi HF, Liu LH, Yu YX, Zi DY, Li ZX et al (1989) Isolationg of Chikungunya virus from bat in Yunnan Province and serological investigations. Chin J Virol 5(1):31–36

    Google Scholar 

  12. Wang JL, Zhang HL, Sun XH, Fu SH, Wang HQ, Feng Y et al (2011) Distribution of mosquitoes and mosquito-borne arboviruses in Yunnan Province near the China-Myanmar-Laos border. Am J Trop Med Hyg 84(5):738–746

    Article  PubMed Central  PubMed  Google Scholar 

  13. Zhang HL, Tao SJ, Yang DR, Zhang YZ, Yang WH, Zhang YZ et al (2005) Isolation of Sindbis, Batai and Coltivirus in Yunnan Province. Chin J Zoonoses 21(7):548–557

    CAS  Google Scholar 

  14. Feng Y, Fu SH, Zhang HL, Li MH, Zhou T, Wang JL et al (2012) Distribution of mosquitoes and mosquito-borne viruses in Yunnan Province along the border of China and Myanmar. Jpn J Infect Dis 65(3):215–221

    Article  PubMed  Google Scholar 

  15. Zhang HL, Zhang YZ, Yang WH, Feng Y, Nasci RS, Yang J et al (2013) Mosquitoes of Western Yunnan Province, China: seasonal abundance, diversity, and arbovirus associations. PLoS One 8(10):e77017

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Attoui H, Jaafar FM, Belhouchet M, Aldrovandi N, Tao SJ, Chen BQ et al (2005) Yunnan orbivirus, a new orbivirus species isolated from Culex tritaeniorhynchus mosquitoes in China. J Gen Virol 86(12):3409–3417

    Article  CAS  PubMed  Google Scholar 

  17. Wang J, Li M, Liang G (2013) Complete genome sequence Analysis of Oya virus, a Simbu serogroup virus of the genus Bunyavirus, newly isolated from China. GenBank.

  18. Kono Y, Yusnita Y, MohdAli AR, Maizan M, Sharifah SH, Fauzia O (2002) Characterization and identification of Oya virus, a Simbu serogroup virus of the genus Bunyavirus, isolated from a pig suspected of Nipah virus infection. Arch Virol 147(8):1623–1630

    Article  CAS  PubMed  Google Scholar 

  19. International Committee on Taxonomy of Viruses (2014) Manzanilla virus, Orthobunyavirus, Bunyaviridae. Virus Taxonomy: 2014 release.

Download references

Authors’ contributions

HLZ and GDL conceived and designed the experiments. YF, BH, SHF and HLZ performed the experiments. WHY, YZZ, YF and HLZ collected mosquitoes. YF, HLZ, GDL, CCT analyzed the data. YF and HLZ wrote the paper. All authors read and approved the final manuscript.


We thank Yong-Hua Liu, Xiao-Xiong Yin and Zheng-Liu Yin from the Ruili City Center for Disease Control and Prevention for collecting mosquitoes. This work was supported by grants from the National Natural Science Foundation of China-Yunnan Province Joint Fund (U1036601), the National Natural Science Foundation of China (81290342), the Ministry of Science and Technology, China (2011CB504702) and the Development Grant of State Key Laboratory for Infectious Disease Prevention and Control (2014SKLID103). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Compliance with ethical guidelines

Competing interests The authors declare that they have no competing interests.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Hai-Lin Zhang.

Additional information

Yun Feng and Shi-Hong Fu contributed equally

Additional files

Additional file 1:

Table s1. Specific primers for Manzanilla virus used in this study.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Feng, Y., Fu, SH., Yang, WH. et al. Isolation and full-length genome analysis of mosquito-borne Manzanilla virus from Yunnan Province, China. BMC Res Notes 8, 255 (2015).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: